The work herein was supported by grants from the United States Government. The United States Government may have certain rights in the invention.
This invention relates to the use of a single bidirectional promoter from the aldehyde reductase gene to regulate expression of two separate genes linked in opposite orientations. More particularly it relates to the use of said bidirectional promoter to regulate expression of two separate genes for applications which require production of two separate gene products in the same cell, including applications which require approximately equimolar amounts of said two separate gene products.
Aldehyde reductase is a member of the aldo-keto reductase superfamily which consists of more than nearly 80 known enzymes and proteins. In addition, genome sequencing projects have identified approximately 150 potential aldo-keto reductase genes, many of which have no assigned function. It is an enzyme which protects cells from environmental and nutritional toxins and carcinogens by detoxification of reactive aldehydes (Bachur, 1976; Feather et al., 1995; Suzuki et al., 1998). It reduces a wide variety of aldehydes to their corresponding alcohols using NADPH as a cofactor. The crystal structures of several members of the superfamily show an xcex1/xcex2 barrel structure with an active site located at the C-terminal end of the barrel (Rondeau et al., 1992; Wilson et al., 1992; Harrison et al., 1994; el-Kabbani et al., 1995; Bennett et al., 1996; Wilson et al., 1995). The catalytic residues are highly conserved in the family and include a Tyr-Lys-Asp catalytic triad (Bohren et al., 1994; Barski et al., 1995; Bruce et al., 1994). The enzymatic properties of this enzyme have been extensively studied, (Barski et al., 1996a; Barski et al., 1996b), and it is ubiquitously distributed in tissues with the highest amounts found in kidney, liver and thyroid (Gabbay et al., 1974; Wirth et al., 1985; Barski et al., 1999). The enzyme is found in all eukaryotes from yeast to mammals. Based on their wide substrate specificity, aldo-keto reductases are thought to be involved in general detoxification of reactive aldehydes. More specific roles for aldehyde reductase were suggested in glucuronate metabolism (Mano et al., 1961), in neurotransmitter metabolism (products of monoamine oxidase) (Tipton et al., 1977; Turner et al., 1974), and in steroid metabolism particularly with respect to isocorticosteroids which are the best-known substrates for this enzyme (Wermuth et al., 1983).
Several regulatory pathways have been identified for the expression of various members of the aldo-keto reductase superfamily. For instance, aldose reductase expression is induced by hypertonicity in all tissues (Bagnasco et al., 1987); and chlordecone reductase is induced in the liver by chlordecone (Molowa et al., 1986a; Molowa et al., 1986b). Comparison of the human aldehyde reductase gene structure to other determined aldo-keto reductase genes (Graham et al., 1991; Wang et al., 1993; Lou et al., 1994; Qin et al., 1994; Khanna et al., 1995) suggests that it is more distantly related to these genes than they are among themselves. Previous studies have not addressed the mechanism of regulation of the aldehyde reductase gene. The present invention identifies alternative splicing of a single primary transcript and the single most important active promoter element. More importantly, the present invention describes the bidirectional function of the promoter of aldehyde reductase and its use to produce two proteins within the same cell.
Expression of two proteins at a time is becoming an increasingly important practical task for research and biotechnology. In biomedicine, it is often necessary to introduce two proteins into the patient""s cells to achieve a therapeutic effect. Examples include two polypeptide chains that assemble into active protein, e.g. light and heavy chain of antibodies, IL-12, etc. New systems for expression of two genes in equal amounts would therefore be useful for production of heterodimeric proteins and for simultaneous expression of selectable or marker genes and a gene of interest.
Examples of other promoters possessing bidirectional activity have been described, yet many of these promoters exhibit significant differences when comparing the transcriptional levels generated by the alternate orientations of the promoter (e.g. Xu et al., 1997; Johnson and Friedmann, 1990 and U.S. Pat. Nos. 5,258,294; 5,338,679; and 5,368,855). Furthermore, there are examples in the art of promoters concluded to be bidirectional (e.g. Koller et al., 1991; Linton et al., 1989); however, in these examples assays for potential bidirectional promoter activity utilized a reporter gene linked to opposite orientations of the promoter on separate constructs.
Other art exists which utilizes the tetracycline (Tet) operon in which operator sequences, regulated by a recombinant Tet repressor, are flanked by two minimal promoters which drive transcription of two separate genes (U.S. Pat. Nos. 5,589,362; 5,654,168; 5,789,156; 5,814,618; 5,866,755; 5,891,718; Clontech). In the Tet system, the same promoter is cloned twice in opposite orientations and drives transcription of separate genes. However, the likelihood of reduced stability of nucleotide sequences increases upon duplication of sequences. Furthermore, at least one operator sequence is present between the two promoters for regulation by the Tet repressor protein. Finally, the Tet system requires the use of specially prepared cell lines which express the regulator protein.
Alternative methods to produce two proteins within the same cell employ variations of constructs which contain an Internal Ribosome Entry Site (IRES) (WO 97/20935; WO 98/11241; WO 98/12338; WO 98/37189; WO 98/49334; WO 98/54342; WO 98/55636; WO 99/24596; WO 99/25817; for specific examples see e.g. U.S. Pat. Nos 5,665,567 or 5,770,428; Clontech), which directs cap-independent translation (Jang et al., 1990; Belsham and Sonenberg, 1996). As a result, one bicistronic RNA is transcribed and two separate proteins are translated from a single transcript. However, this method has an inherent drawback that translation from an IRES is often reduced compared to the initial translation start site (Jang et al., 1989). Another alternative is to use two promoters, one for each gene. However, this may lead to construct instability or to grossly different levels of transcription (Ju et al., 1980; Junker et al., 1995). Using a single bidirectional promoter provides a potential for expression of two genes in equivalent amounts and may circumvent drawbacks of current alternatives.
An embodiment of the present invention is a DNA sequence and fragments and derivative thereofwhich function as bidirectional promoters. An additional embodiment is said DNA sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29; SEQ ID NO:30 and SEQ ID NO:31. In a specific embodiment, said promoter has the characteristic of promoting transcription of two separate nucleotide sequences wherein one of such nucleotide sequences is operatively linked to the 5xe2x80x2 end of said promoter sequence and is transcribed 5xe2x80x2 to 3xe2x80x2 in the direction opposite from the 5xe2x80x2 to 3xe2x80x2 direction of said promoter sequence and the other of such nucleotide sequences is operatively linked to the 3xe2x80x2 end of said promoter sequence and is transcribed 5xe2x80x2 to 3xe2x80x2 in the same direction as the 5xe2x80x2 to 3xe2x80x2 direction of said promoter sequence. In a further embodiment, said promoter promotes transcription of the said two nucleotide sequences in approximately equimolar amounts. In an additional embodiment said promoter is the aldehyde reductase promoter.
In another embodiment of the present invention there is a recombinant DNA vector comprising the promoter sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29; SEQ ID NO:30 and SEQ ID NO:31 or fragments and derivatives thereof, wherein said fragments and derivatives function as a bidirectional promoter; a first DNA sequence encoding a gene operatively linked to the 5xe2x80x2 end of the promoter sequence wherein when said first DNA sequence is transcribed it is transcribed 5xe2x80x2 to 3xe2x80x2 in the direction opposite from the 5xe2x80x2 to 3xe2x80x2 direction of the promoter sequence; and a second DNA sequence encoding a gene operatively linked to the 3xe2x80x2 end of the promoter sequence wherein when said second DNA sequence is transcribed it is transcribed 5xe2x80x2 to 3xe2x80x2 in the same direction as the 5xe2x80x2 to 3xe2x80x2 direction of the promoter sequence. In a specific embodiment, said vector also contains a poly A+ polyadenylation sequence operatively linked to the 3xe2x80x2 end of at least one of said first or second nucleotide sequences. In a further embodiment, said nucleotide sequences are nonidentical. In a specific embodiment, at least one said nucleotide sequence of said vector encodes a reporter sequence. In a specific embodiment said reporter sequence is selected from the group consisting of ampicillin, neomycin, kanamycin, luciferase, xcex2-galactosidase, xcex2-glucuronidase, chloramphenicol acetyl transferase (CAT), blue fluorescent protein (BFP), green fluorescent protein (GFP), or placental alkaline phosphatase. In further embodiments, said nucleotide sequences encode heavy and light chains of an antibody, subunits of an interleukin, subunits of a growth hormone receptor, a subunit of a homodimer, or subunits of a heteterodimer. In an additional embodiment we claim the method of preparing said antibody, interleukin, growth hormone receptor, homodimer, and heterodimer comprising expression of said nucleotide sequences and recovering the formed product. A specific embodiment of the present invention is the vector wherein one DNA sequence encodes a therapeutic nucleotide sequence and the other DNA sequence encodes a suicide gene.
An additional embodiment of the present invention is said vector wherein at least one nucleotide sequence encodes an RNA; said RNA being the final product of said nucleotide sequence and containing functional characteristics. In another embodiment we claim the method of preparing a recombinant ribonucleoprotein encoded for by the DNA sequences of the recombinant DNA vector comprising the expression of said DNA sequences and recovery of the recombinant functional ribonucleoprotein. In a further embodiment we claim the method of preparing recombinant RNAs encoded for by the sequences of said recombinant DNA vector comprising expression of said DNA sequences and recovery of functional RNAs. In specific embodiments said functional RNA can be a snRNA, a snoRNA, a scRNA, the telomerase RNA, an antisense RNA, or the XIST RNA.
An additional embodiment is the recombinant DNA vector comprising the promoter sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29; SEQ ID NO:30 and SEQ ID NO:31 or fragments and derivatives thereof when said fragments and derivatives function as a bidirectional promoter; a first polylinker site into which a first DNA sequence is operatively linked to the 5xe2x80x2 end of the promoter sequence wherein when said first DNA sequence is transcribed it is transcribed 5xe2x80x2 to 3xe2x80x2 in the direction opposite from the 5xe2x80x2 to 3xe2x80x2 direction of the promoter sequence; and a second polylinker site into which a second DNA sequence encoding a gene operatively linked to the 3xe2x80x2 end of the promoter sequence wherein when said second DNA sequence is transcribed it is transcribed 5xe2x80x2 to 3xe2x80x2 in the same direction as the 5xe2x80x2 to 3xe2x80x2 direction of the promoter sequence.
An additional embodiment is said vector further comprising a poly A+ polyadenylation sequence operatively linked to the 3xe2x80x2 end of at least one of said first or second nucleotide sequences.
In a specific embodiment there is a recombinant DNA vector comprising a cassette, wherein said cassette comprises one DNA sequence which encodes a suicide nucleic acid sequence and the other DNA sequence which encodes an immortalization nucleic acid sequence. In a specific embodiment the suicide nucleic acid sequence is thymidine kinase (TK). In another specific embodiment the immortalization nucleic acid sequence is selected from the group consisting of T-antigen, telomerase catalytic protein subunit and myc.
In another specific embodiment there is a method to initiate proliferation of a cell comprising the step of introducing the vector comprising a cassette, wherein said cassette comprises one DNA sequence which encodes a suicide nucleic acid sequence and the other DNA sequence which encodes an immortalization nucleic acid sequence, into said cell under conditions wherein activation of the bidirectional promoter to transcribe said immortalization nucleic acid sequence in said cell initiates proliferation of said cell. In another specific embodiment the vector further comprises excision sites flanking said cassette, wherein said excision sites are selected from the group consisting of lox, FLP recognition target sites, restriction endonuclease sites, and transposon sequences.
In an additional specific embodiment there is a method to initiate proliferation of a cell comprising the step of introducing a vector comprising a cassette, wherein said cassette comprises one DNA sequence which encodes a suicide nucleic acid sequence and the other DNA sequence which encodes an immortalization nucleic acid sequence, wherein the vector further comprises excision sites flanking said cassette, wherein said excision sites are selected from the group consisting of lox, FLP recognition target sites, restriction endonuclease sites, and transposon sequences, into said cell under conditions wherein activation of the bidirectional promoter to transcribe said immortalization nucleic acid sequence in said cell initiates proliferation of said cell. In a specific embodiment the introduction step further comprises integration of said excision sites and said cassette into a provirus of said cell. In an additional embodiment the method further comprises excising said immortalization nucleic acid sequence from said provirus through said excision sites. In a further specific embodiment the method further comprises destroying a cell which has failed to excise said immortalization nucleic acid sequence. In an additional specific embodiment the cell is a primary cell. In another specific embodiment the primary cell is selected from the group consisting of insulin-producing beta cell and liver cell.